Method of treating pregnant cows and/or heifers

ABSTRACT

The present invention relates to combination vaccines for the prophylaxis and treatment of microbiological infections in cattle which comprise an attenuated bovine viral diarrhea virus (BVDV) for the the prophylaxis and treatment of BVDV caused infections. The invention also relates to a method of vaccinating a pregnant cow.

SEQUENCE LISTING

This application contains a sequence listing in computer readable format, the teachings and content of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the field of animal health and in particular to vaccines which comprise an attenuated bovine viral diarrhea virus (BVDV). More particularly, the present invention relates to the use of modified live BVDV Type 1 and BVDV Type 2 vaccines. Still more particularly, the present invention relates to the use of such vaccines in pregnant cows/heifers. Even more particularly, the vaccines will include other antigens from bovine pathogens in addition to the BVDV Type 1 and BVDV Type 2 modified live vaccines.

BACKGROUND OF THE INVENTION

Cows are susceptible to contracting a large diversity of microbial infections. A number of effective vaccines have been developed to treat or prevent infection. Vaccines that are based on modified live viruses, however, pose a risk to the health of pregnant cows and their calves. Accordingly, there is a need for safe methods of vaccinating pregnant cows.

Bovine viral diarrhea virus (BVDV) Type 1 (BVDV-1) and Type 2 (BVDV-2) cause bovine viral diarrhea (BVD) and mucosal disease (MD) in cattle, as well as cause abortions and fetal infections that may result in persistently infected calves (Baker, 1987; Moennig and Plagemann, 1992;). The division of BVDV into 2 species is based on significant differences at the level of genomic sequences (summarized in Heinz et al., 2000) which are also obvious from limited cross neutralizing antibody reactions. The viral proteins of BVDV, and any other virus of the pestivirus family, are arranged in the polyprotein in the order NH₂-N^(pro)-C-E^(rns)-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (Lindenbach and Rice, 2001).

Present BVDV vaccines for the prevention and treatment of BVDV infections still have drawbacks. Vaccines against the classical BVDV-1 provide only partial protection from BVDV-2 infection, and vaccinated dams may produce calves that are persistently infected with virulent BVDV-2. This problem is probably due to the great antigenic diversity between type 1 and type 2 strains which is most pronounced in the glycoprotein E2, the major antigen for virus neutralization. Most monoclonal antibodies against type 1 strains fail to bind to type 2 viruses.

Currently, licensed BVDV MLV vaccines are produced using attenuated viruses obtained via repeated passage in bovine or porcine cells, or using chemically modified viruses which exhibit a temperature-sensitive phenotype. A single dose of MLV vaccine is sufficient for immunization, and duration of the immunity can last for years in vaccinated cattle. However, these vaccines, although attenuated, are often associated with safety problems. Prior art has taught that the vaccine viruses may cross the placenta of pregnant animals, e.g. cows or heifers and lead to clinical manifestations in the fetus and/or the induction of persistently infected calves. Therefore, it is recommended that they not be applied or administered to breeding herds that contain pregnant cows. Pregnant cows have to be kept separate from vaccinated cattle to protect fetuses and must not be vaccinated themselves.

Bovine Herpesvirus (BHV-1) or Infectious bovine rhinotracheitis (IBR) virus is associated with several diseases and symptoms in cattle: Infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV), balanoposthitis, conjunctivitis, abortion, encephalomyelitis, and mastitis. Only a single serotype of BHV-1 is recognized; however, three subtypes of BHV-1 have been described on the basis of endonuclease cleavage patterns of viral DNA. These types are referred to as BHV-1.1 (respiratory subtype), BHV-1.2 (genital subtype), and BHV-1.3 (encephalitic subtype). Recently, BHV-1.3 has been reclassified as a distinct herpesvirus designated BHV-5. BHV-1 infections are widespread in the cattle population. In feedlot cattle, the respiratory form is most common. The viral infection alone is not life-threatening but predisposes cattle to secondary bacterial pneumonia, which may result in death. In breeding cattle, abortion or genital infections are more common. Genital infections can occur in bulls (infectious pustular balanoposthitis) and cows (IPV) within 1-3 days of mating or close contact with an infected animal. Transmission can occur in the absence of visible lesions and through artificial insemination with semen from subclinically infected bulls. Cattle with latent BHV-1 infections generally show no clinical signs when the virus is reactivated, but they do serve as a source of infection for other susceptible animals and thus perpetuate the disease. The incubation period for the respiratory and genital forms is 2-6 days. In the respiratory form, clinical signs range from mild to severe, depending on the presence of secondary bacterial pneumonia. Clinical signs include pyrexia, anorexia, coughing, excessive salivation, nasal discharge that progresses from serous to mucopurulent, conjunctivitis with lacrimal discharge, inflamed nares (hence the common name “red nose”), and dyspnea if the larynx becomes occluded with purulent material. Pustules may develop on the nasal mucosa and later form diphtheritic plaques. Conjunctivitis with corneal opacity may develop as the only manifestation of BHV-1 infection. In the absence of bacterial pneumonia, recovery generally occurs 4-5 days after the onset of clinical signs. Abortions may occur concurrently with respiratory disease but can also occur up to 100 days after infection. Abortions can occur regardless of the severity of disease in the dam. Abortions generally occur during the second half of pregnancy, but early embryonic death may also occur. The first signs of genital infections in cows are frequent urination, elevation of the tailhead, and a mild vaginal discharge. The vulva is swollen, and small papules, then erosions and ulcers, are present on the mucosal surface. If secondary bacterial infections do not occur, animals recover in 10-14 days. If bacterial infection occurs, there may be inflammation of the uterus and transient infertility, with purulent vaginal discharge for several weeks. In bulls, similar lesions occur on the penis and prepuce. BHV-1 infection can be severe in young calves and cause a generalized disease. Pyrexia, ocular and nasal discharges, respiratory distress, diarrhea, incoordination, and eventually convulsions and death may occur in a short period after generalized viral infection. IBR is rarely fatal in cattle unless complicated by bacterial pneumonia. In uncomplicated IBR infections, most lesions are restricted to the upper respiratory tract and trachea. Petechial to ecchymotic hemorrhages may be found in the mucous membranes of the nasal cavity and the paranasal sinuses. Focal areas of necrosis develop in the nose, pharynx, larynx, and trachea. The lesions may coalesce to form plaques. The sinuses are often filled with a serous or serofibrinous exudate. As the disease progresses, the pharynx becomes covered with a serofibrinous exudate, and blood-tinged fluid may be found in the trachea. The pharyngeal and pulmonary lymph nodes may be acutely swollen and hemorrhagic. The tracheitis may extend into the bronchi and bronchioles; when this occurs, epithelium is sloughed in the airways. The viral lesions are often masked by secondary bacterial infections. In young animals with generalized BHV-1 infection, erosions and ulcers overlaid with debris may be found in the nose, esophagus, and forestomachs. In addition, white foci may be found in the liver, kidney, spleen, and lymph nodes. Aborted fetuses may have pale, focal, necrotic lesions in all tissues, but which are especially visible in the liver.

Parainfluenza-3 virus (PI-3) is an RNA virus classified in the paramyxovirus family. Infections caused by PI-3 are common in cattle. Although PI-3 is capable of causing disease, it is usually associated with mild to subclinical infections. The most important role of PI-3 is to serve as an initiator that can lead to the development of secondary bacterial pneumonia. Clinical signs include pyrexia, cough, serous nasal and lacrimal discharge, increased respiratory rate, and increased breath sounds. The severity of signs worsen with the onset of bacterial pneumonia. Fatalities from uncomplicated PI-3 pneumonia are rare. Lesions include cranioventral lung consolidation, bronchiolitis, and alveolitis with marked congestion and hemorrhage. Inclusion bodies may be identified. Most fatal cases will also have a concurrent bacterial bronchopneumonia.

Bovine Respiratory Syncytial Virus (BRSV) is an RNA virus classified as a pneumovirus in the paramyxovirus family. In addition to cattle, sheep and goats can also be infected by respiratory syncytial viruses. This virus was named for its characteristic cytopathic effect—the formation of syncytial cells. Antigenic subtypes are known to exist for BRSV, and preliminary evidence suggests that there may be antigenic subtypes of BRSV. BRSV is distributed worldwide, and the virus is indigenous in the cattle population. BRSV infections associated with respiratory disease occur predominantly in young beef and dairy cattle. Passively derived immunity does not appear to prevent BRSV infections but will reduce the severity of disease. Initial exposures to the virus are associated with severe respiratory disease; subsequent exposures result in mild to subclinical disease. BRSV appears to be an important virus in the bovine respiratory disease complex because of its frequency of occurrence, predilection for the lower respiratory tract, and its ability to predispose the respiratory tract to secondary bacterial infection. In outbreaks, morbidity tends to be high, and case fatality can be 0-20%. Signs include increased rectal temperature (40-42° C.), depression, decreased feed intake, increased respiratory rate, cough, and nasal and lacrimal discharge. Generally, respiratory signs predominate. Dyspnea may become pronounced in the later stages of the disease. Subcutaneous emphysema is sometimes reported. Secondary bacterial pneumonia is a frequent occurrence. A biphasic disease pattern has been described but is not consistent. Gross lesions include a diffuse interstitial pneumonia with subpleural and interstitial emphysema along with interstitial edema. These lesions are similar to and must be differentiated from other causes of interstitial pneumonia. See also atypical interstitial pneumonia. Histologic examination reveals syncytial cells in bronchiolar epithelium and lung parenchyma, intracytoplasmic inclusion bodies, proliferation and/or degeneration of bronchiolar epithelium, alveolar epithelialization, edema, and hyaline membrane formation.

Leptospirosis is a contagious disease of animals, including man, caused by various immunologically distinct leptospiral serovars, most of which are regarded as subgroups of Leptospira interrogans. Infections may be asymptomatic or cause various signs, including fever, icterus, hemoglobinuria, renal failure, infertility, abortion, and death. After acute infection, leptospires frequently localize in the kidneys or reproductive organs and are shed in the urine, sometimes in large numbers for months or years. Because the organisms survive in surface waters for extended periods, the disease is often waterborne. In the USA, the disease is primarily due to the serovars Leptospira hardjo, Leptospira pomona, and Leptospira grippotyphosa. However, Leptospira canicola and icterohaemorrhagiae serovars also have been isolated. Calves may have fever, anorexia, and dyspnea, and in Leptospira pomona infections, icterus, hemoglobinuria, and anemia. Body temperature may rise suddenly to 40.5-41° C. Hemoglobinuria rarely lasts longer than 48-72 hrs. Icterus clears rapidly and is followed by anemia. The RBC's begin to increase in number by 4-5 days and return to normal 7-10 days later. However, Leptospira hardjo infections usually do not cause hemolytic anemia, which makes diagnosis more difficult. Morbidity and mortality are higher in calves than in adult cattle. In older cattle, signs vary greatly and diagnosis is more difficult. Enzootic Leptospira hardjo infections, which usually result in abnormal milk, are more obvious in dairy than in beef cattle. Signs usually are restricted to lowered milk and calf production; a hemolytic crisis does not occur. The milk is thick, yellow, and blood-tinged; it may contain clots, although there is little evidence of mammary inflammation. Milk production returns to normal in 10-14 days, even in the absence of treatment. Abortion and stillbirths, which are common in Leptospira pomona infections and sporadic in Leptospira hardjo infections, generally occur 3-10 weeks after initial infection. The abortions are more common during the third trimester. An abortion storm in a breeding herd is often the first indication that leptospirosis exists, because the mild initial signs often pass unnoticed. In endemically infected herds, abortions occur mostly in younger animals and are sporadic, rather than being manifested as abortion storms. Calves reared by previously infected cows are protected by colostral antibodies for up to 6 mos. The calves generally have an antibody titer similar to that of their dams. In the acute form, anemia, icterus, hemoglobinuria, and submucosal hemorrhages are prominent. The kidneys are swollen, with multifocal petechial and ecchymotic hemorrhages that become pale with time. The liver may be swollen, with minute areas of focal necrosis. Petechiae in other organs are seen in fulminating cases; however, in the more prevalent Leptospira hardjo infections, the lesions are primarily restricted to the kidneys.

Haemophilus somnus is being increasingly recognized as an important pathogen in BRD; these bacteria are normal inhabitants of the nasopharynx of cattle. H. somnus infection of the lungs results in purulent bronchopneumonia that may be followed by septicemia and infection of multiple organs. Occasionally, H. somnus is associated with extensive pleuritis. H. somnus can cause an acute, usually fatal, septicemic disease that can involve the nervous, musculoskeletal, circulatory, and respiratory systems, either singly or together. The reproductive system is often affected but usually without the other systems being clinically involved. The disease may be characterized by fever, severe depression, ataxia, weakness, blindness, coma, and death within several hours to several days. It occurs sporadically in individual beef and dairy cattle and is found nearly worldwide. H. somnus is a gram-negative, nonmotile, nonsporeforming, pleomorphic coccobacillus that requires an enriched medium and a microaerophilic atmosphere for culture.

DESCRIPTION OF THE INVENTION

One aspect of the present invention provides a method for safely vaccinating a pregnant cow, wherein the method generally includes or comprises the step of administering a vaccine to the pregnant cow or pregnant heifer. The vaccine preferably includes an immunological active component effective for treating or prophylaxis of an infection caused by a microbe selected from the group consisting of: Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhea Virus (BVDV) Type 1, BVDV Type 2, Parainfluenza-3 Virus (PI-3), Bovine Respiratory Syncytial Virus (BRSV), and combinations thereof. The pregnant cow or heifer can be in the first, second, or third trimester of pregnancy when the vaccine is administered. In preferred forms of this vaccine, the immunological active components effective for treating or prophylaxis of infection caused by BVDV Type 1 and/or BVDV Type 2 is a modified live BVDV. In other preferred forms, the immunological active component effective for treating or prophylaxis of infection caused by IBR is a modified live IBR. Preferred vaccine compositions will include both BVDV Type 1 and BVDV Type 2 immunological active components, even more preferably both the BVDV Type 1 and BVDV Type 2 immunological active components will be modified live BVDV, still more preferably, preferred vaccine compositions will further include IBR immunological active components, preferably modified live IBR. In even further preferred vaccine compositions, PI-3 immunological active components will be included. In still further preferred vaccine compositions, BRSV immunological active components will be included. Representative vaccines that can be used in a method according to the invention including modified live BVDV Type I, modified live BVDV Type II, modified live IBR, PI-3, and BRSV that are sold under the names BREED-BACK™ FP5, EXPRESS™ FP5, and EXPRESS™ 5 (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Mo.).

According to a further aspect the present invention relates to the vaccination of pregnant cows/heifers comprising the step of administering to a pregnant cow/heifer a modified live BVDV Type 1, preferably a cytopathic modified lived BVDV Type 1, which is derived from the Singer strain, a modified live BVDV Type 2, preferably a cytopathic modified live BVDV Type 2, which is derived from the 296 strain, and/or a modified live IBR which is derived from the IBR Colorado 1 Strain. Preferably the modified live BVDV Type 1 Singer strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live BVDV Type 2 296 strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live IBR strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the vaccine is administered to prevent, or reduce the incidence of or severity of an infection in the pregnant cow and preferably its fetus with or at least the clinical signs caused by BVDV Type 1, BVDV Type 2, and/or IBR (each depending from the antigen/antigen combination that is/are used—modified live BVDV Type I, modified live BVDV Type 2, modified live IBR or any combination thereof).

In other aspect the present invention also relates to a method for vaccinating pregnant cows/heifers comprising the step of administering to said pregnant cow/heifer a vaccine which comprises a vaccine composition and/or immunological active components of Haemophilus somnus, preferably in a bacterin form, in addition to the modified live BVDV Type 1, modified live BVDV Type 2, and/or modified live IBR, described above. Preferably said vaccine further comprises PI-3 and BRSV immunological active components. Representative vaccines including modified live BVDV Type 1, modified live BVDV Type 2, modified live IBR, PI-3, BRSV, and Haemophilus somnus bacterin that can be used for the vaccination of pregnant cows as described herein are sold under the names BREED-BACK™ FP5-HS and EXPRESS™ 5-HS (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Mo.).

According to a further aspect, the vaccine composition that can be used for the vaccination of pregnant cows/heifers as described herein further comprises a vaccine composition or immunological active component effective for treating or preventing an infection caused by a Leptospira bacterium. In preferred forms of such vaccine composition, the immunological active component will be a Leptospira bacterin. Still more preferably, the bacterin will be derived from a Leptospira bacterium selected from the group consisting of: Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, Leptospira Pomona, and combinations thereof. Some preferred vaccine compositions will include Leptospira bacterin derived from Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. Representative vaccines including modified live BVDV Type 1, modified live BVDV Type 2, modified live IBR, PI-3, BRSV, Haemophilus somnus bacterin, and Leptospira bacterin derived from Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona that can be used for the vaccination of pregnant cows/heifers as described herein are sold under the names BREED-BACK™ FP10-HS and EXPRESS 10-HS® (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Mo). Representative vaccines including modified live BVDV Type 1, modified live BVDV Type 2, modified live IBR, PI-3, BRSV, and Leptospira bacterin derived from Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona that can be used for the vaccination of pregnant cows/heifers as described herein are sold under the names BREED-BACK™ FP10 and EXPRESS™10 (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, Mo.).

According to a further aspect, the vaccine composition that can be used for the vaccination of pregnant cows/heifers as described herein further comprises at least one further immunolical active component which can prevent the pregnant cow/heifer from microbiological infections of other cattle relevant pathogens or at least from the clinical signs caused by said other cattle relevant pathogens. A list of those pathogens and vaccine compositions which are also suitable to be used according to the present invention are disclosed in the international patent application published as WO 2007-117303, the entire teachings and content of which is incorporated herein by reference.

For all of the vaccine compositions described above and that can be used for the vaccination of pregnant cows/heifers as described herein, the modified live viruses used therein are preferably attenuated. A particularly preferred modified live BVDV Type 1 is derived from a Singer strain of BVDV. Even more preferably, the Singer strain of BVDV has been passaged at least two times in bovine turbinate cells. Most preferably the modified BVDV Singer strain is the one that is included in the BREED-BACK™ FP5 vaccine. A particularly preferred modified live BVD Type 2 is derived from BVDV Strain 296. Even more preferably, the BVDV Strain 296 has been passaged in bovine testicular cells or MDBK cells, or embryonic swine kidney cells (ESK cells). In particularly preferred forms, the BVDV Strain 296 has been passaged in bovine testicular cells at least six times and in MDBK and ESK cells at least 23 times. Most preferably the modified BVDV 296 strain is the one that is included in the BREED-BACK™ FP5 vaccine.

A particularly preferred modified live IBR strain is derived from the IBR Colorado 1 strain. Even more preferably, the IBR Colorado 1 Strain 1 has been passaged 4 times in bovine embryo kidney cells, 22 times in ovine cells and once in MDBK cells. Most preferably the modified IBR strain is the one that is included in the BREED-BACK™ FP5 vaccine. In other preferred forms of practicing the invention, in addition to the administration of any vaccine composition described above to a pregnant cow or heifer, the vaccine composition is also administered to the cow prior to the pregnancy. Preferably, such administration is performed within 12 months of the pregnancy, still more preferably within 10 months, even more preferably within 8 months, still more preferably within 7 months, even more preferably within 6 months, still more preferably within 5 months, even more preferably within 4 months, still more preferably within 3 months, even more preferably within 2 months, and most preferably between about 1 and two months prior to pregnancy.

In some preferred forms of any of the vaccine compositions described above, the composition will include at least one additional component selected from the group consisting of pharmaceutical acceptable carriers, adjuvants, diluents, preservatives, antibiotics, and combinations thereof.

“Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene oil resulting from theoligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly appropriate for compositions containing such adjuvants. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

Additionally, the composition can include one or more pharmaceutical-acceptable carriers. As used herein, “a pharmaceutical-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkalisalts of ethylendiamintetracetic acid, among others.

A “protectant” or “preservative” as used herein, refers to an anti-microbiological active agent, such as for example Neomycin, Amphoteracin B, Gentamycin, Merthiolate, and the like. Of these, Neomycin and/or Amphoteracin B are particularly preferred In particular adding a protectant is most preferred for the preparation of a multi-dose composition. Those anti-microbiological active agents are added in concentrations effective to prevent the composition of interest from any microbiological contamination or for inhibition of any microbiological growth within the composition of interest.

Moreover, this method can also comprise the addition of any stabilizing agent, such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and/or maintain product shelf-life.

In preferred forms, administration of any vaccine composition noted above will be safe for the animal receiving the composition both before and during pregnancy. “Safe” as used herein, shall refer to a comparison in animals receiving the composition with animals not receiving the composition wherein the abortion rates of the vaccinated pregnant cows/heifers are less than 10% higher, more preferably less than 5% higher, still more preferably less than 4% higher, even more preferably less than 3% higher, still more preferably less than 2% higher, even more preferably less than 1% higher, and most preferably no higher than unvaccinated cows/heifers. Additionally, administration of any vaccine composition noted above to a pregnant cow/heifer will not cause fetal infection by any pathogen having an immunological active component in the vaccine in more than 10%, more preferably 5%, still more preferably 4%, even more preferably 3%, still more preferably 2%, even more preferably 1% and most preferably no higher than unvaccinated cows/heifers.

Thus according a further aspect the present invention also relates to method for the treatment or prophylaxis of a pregnant cow/heifer and its fetus against infections, or at least the clinical signs, caused by caused by BVDV comprising the step administering to said pregnant cow/heifer a vaccine comprising modified live live bovine viral diarrhea virus as described above. Preferably the live bovine viral diarrhea virus is a modified live BVDV Type 1 strain derived from the Singer strain and/or a modified live BVDV Type 2 derived from the 296 strain. Preferably the modified live BVDV Type 1 Singer strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live BVDV Type 2 296 strain is the one that is included in the BREED-BACK™ FP5 vaccine. Representative vaccines are also sold under the names BREED-BACK™ FP5-HS, EXPRESS™ 5-HS, BREED-BACK™ FP10-HS and EXPRESS 10-HS®.

According a further aspect, the present invention also relates to a method for the treatment or prophylaxis of pregnant cow/heifer and its fetus against infections, or at least the clinical signs, caused by IBR comprising the step administering to said pregnant cow/heifer a vaccine comprising modified live live IBR as described above. Preferably the modified IBR strain is the one that is included in the BREED-BACK™ FP5 vaccine. Representative vaccines are also sold under the names BREED-BACK™ FP5-HS, EXPRESS™ 5-HS, BREED-BACK™ FP10-HS and EXPRESS 10-HS®.

Thus according to a further aspect, the present invention also relates to method for the treatment or prophylaxis of a pregnant cow/heifer and its fetus against infections, or at least the clinical signs, caused by BVDV and IBR comprising the step administering to said pregnant cow/heifer a vaccine comprising modified live live bovine viral diarrhea virus and modified live IBR as described above. Preferably the live bovine viral diarrhea virus is a modified live BVDV Type 1 strain derived from the Singer strain and/or a modified live BVDV Type 2 derived from the 296 strain. Preferably the modified live BVDV Type 1 Singer strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live BVDV Type 2 296 strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified IBR strain is the one that is included in the BREED-BACK™ FP5 vaccine. Representative vaccines are also sold under the names BREED-BACK™ FP5-HS, EXPRESS™ FP5, EXPRESS™ 5-HS, BREED-BACK™ FP10-HS and EXPRESS 10-HS®.

In preferred methods of the present invention, pregnant cows/heifers receiving an administration of any of the vaccine compositions described above will experience “treatment or prophylaxis” in the form of a decrease or reduction in the incidence of or severity of clinical, pathological, and histopathological signs of infection from any of the pathogens having an immunological active component included in the administered vaccine. “Decrease” or “reduction in the incidence of or severity of clinical, pathological, and/or histopathological signs” shall mean that clinical signs are reduced in incidence or severity in animals receiving an administration of the vaccine in comparison with a “control group” of animals when both have been infected with or challenged by the pathogen from which the immunological active component(s) in the vaccine are derived and wherein the control group has not received an administration of the vaccine. In this context, the term “decrease” or “reduction” means a reduction of at least 10%, preferably 25%, even more preferably 50%, most preferably of more than 100% in the vaccinated group as compared to the control group as defined above.

“Clinical signs” shall refer to signs of infection from a pathogen that are directly observable from a live animal such as symptoms. Representative examples will depend on the pathogen selected but can include things such as nasal discharge, lethargy, coughing, elevated fever, weight gain or loss, dehydration, diarrhea, swelling, lameness, and the like.

“Pathological” signs shall refer to signs of infection that are observable at the microscopic or molecular level, through biochemical testing, or with the naked eye upon necropsy.

“Histopathological” signs shall refer to signs of tissue changes resulting from infection. Administration of any of the vaccine compositions described herein can occur in any conventional manner or fashion known in the art with intramuscularly and subcutaneously being particularly preferred, and with subcutaneous administration being the most preferred.

In another aspect of the present invention, a method of treatment or prophylaxis of pregnant cows or heifers of clinical signs or symptoms of infection caused by bovine viral diarrhea virus is provided. Generally, the method comprises the step of administering an immunogenic composition to a pregnant cow/heifer, wherein the immunogenic composition comprises a modified live bovine viral diarrhea virus. Preferably the live bovine viral diarrhea virus is a modified live BVDV Type 1 strain derived from the Singer strain and/or a modified live BVDV Type 2 derived from the 296 strain. Preferably the modified live BVDV Type 1 Singer strain is the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live BVDV Type 2 296 strain is the one that is included in the BREED-BACK™ FP5 vaccine. Representative vaccines are also sold under the names BREED-BACK™ FP5-HS, EXPRESS™ 5-HS, BREED-BACK™ FP10-HS and EXPRESS 10-HS®. In preferred forms, the immunogenic composition, routes of administration, timing of administration, and variations noted above in describing the vaccine compositions are the same for the immunogenic composition and methods of this aspect.

In another aspect of the present invention, the vaccine or immunogenic composition as described herein is administered to healthy susceptible cows and replacement heifers prior to breeding wherein such administration prevents persistently infected calves due to BVD Types 1 and 2.

In another aspect of the present invention, the vaccine or immunogenic composition as described herein aids in the reduction of respiratory diseases caused by IBR virus, BVDV Types 1 and 2, PI-3 virus, and BRSV when administered to pregnant females or calves nursing pregnant females that had been vaccinated with the same vaccine or immunogenic composition prior to breeding or being artificially inseminated. In preferred forms of this aspect, for cows and heifers and using aseptic techniques, annually inject a 2 mL dose intramuscularly or subcutaneously at or about four weeks prior to breeding. Pregnant cows and nursing calves may be vaccinated following the pre-breeding vaccination.

For the aspects of the invention described herein, attenuated modified live BVDV Type 1 and 2 strains are grown in MDBK-cells until a TCID₅₀ of about 10^(5.0) to 10^(8.1) per ml cell culture fluid. A modified live attenuated strain of IBR is grown in MDBK cells until a TCID₅₀ of about 10^(5.0) to 10^(8.6) per ml cell culture fluid. A live attenuated strain of BRSV is grown in MDBK cells until a TCID₅₀ of about 10^(5.0) to 10^(7.2) per ml cell culture fluid. A live attenuated strain of PI-3 is grown in MDBK cells until a TCID₅₀ of about 10^(4.2) to 10^(6.5) per ml cell culture fluid. Leptospira bacterium, and especially those described above, are separately cultivated until reaching 10^(8.0) to 10^(11.0) cells per ml culture. The bacteria cultures are inactivated and the culture fluids are lyophilized or freeze dried. H. somnus is cultivated until achieving 10^(7.1) to 10^(9.2) cells per ml culture. The bacteria culture is inactivated and the culture fluid is lyophilized or freeze dried.

For any of the vaccine compositions described herein, final dosage amounts for the individual components is as follows: IBR (10^(5.0) to 10^(8.6) TCID₅₀), BVDV-1 (10^(5.0) to 10^(8.1) TCID₅₀), BVDV-2 (10^(5.0) to 10^(8.1) TCID₅₀), BRSV (10^(5.0) to 10^(7.2) TCID₅₀), PI3 (10^(4.2) to 10^(6.5) TCID₅₀), each selected Leptospira bacteria 10^(8.0) to 10^(11.0) cells per ml culture, and Haemophilus somnus 10^(7.1) to 10^(9.2) cells per ml culture. Still more preferably, the dosage amounts are as found in a product selected from the group consisting of BREED-BACK™ 5, EXPRESS™ 5, BREED-BACK™ FP5, EXPRESS™ FP5, BREED-BACK™ 5HS, EXPRESS™ 5HS, BREED-BACK™ FP5-HS, EXPRESS™ FP5-HS, BREED-BACK™ 10, EXPRESS™ 10, BREED-BACK™ FP10, EXPRESS™ 10, BREEDBACK™ FP10-HS, EXPRESS FP10-HS®, BREED-BACK™ 10HS, EXPRESS™ FP3-VL5, and EXPRESS™ FP5-VL5. It is noted that the BREED-BACK product line has been replaced by the EXPRESS product line. However, this change was in name only as all components therein remained the same.

Further Definitions:

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a BVDV” includes a plurality of such BVDV, reference to the “cell” is a reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties including for the purpose of describing and disclosing the cell lines, vectors, and methodologies as reported in the publications, which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term “BVDV” as used herein refers to all viruses belonging to species bovine viral diarrhea virus (BVDV) type 1 (BVDV-1) and BVDV type 2 (BVDV-2), including any sub-species such as 1a, 1b, 2a, 2,b, and the like in the genus Pestivirus within the family Flaviviridae (Heinz et al., 2000). The more classical BVDV type 1 strains and the more recently recognized BVDV type 2 strains display some limited but distinctive differences in nucleotide and amino acid sequences. Preferably the term BVDV as used herein refers to the modified BVDV. Even more preferably the modified BVDV as used herein refers to a cytopathic modified live BVDV Type 1 strain derived from the Singer strain and/or a cytopathic modified live BVDV Type 2 derived from the 296 strain. Preferably the modified live BVDV Type 1 Singer strain refers to the one that is included in the BREED-BACK™ FP5 vaccine. Preferably the modified live BVDV Type 2 296 strain refers to the one that is included in the BREED-BACK™ FP5 vaccine.

“A modified live BVDV” as used herein means that there is a statistically significant difference between the virulence of modified live BVDV of the present invention, and wild-type BVDV isolates including those from which said modified live BVDV have been derived, for the predominant clinical parameters, in case of BVDV for diarrhea, pyrexia and lethality in animals infected with the same dose, which may be 6×10⁶ TCID₅₀. Thus, said modified live BVDV have a reduced incidence of or severity of clinical, pathological, and histopathological signs associated with BVDV infection, including diarrhea, pyrexia and lethality.

“Immunological active component” or “immunologically active component” as used herein means a component that induces or stimulates the immune response in an animal to which said component is administered. Said immune response may be directed to said component or to a microorganism comprising said component. The immunological active component may be an attenuated microorganism, including modified live virus (MLV), a killed-microorganism or at least an immunological active part of a microorganism.

“Immunological active part of a microorganism” as used herein means a protein-, sugar-, and or glycoprotein containing fraction of a microorganism that comprises at least one antigen that induces or stimulates the immune response in an animal to which said component is administered. Said immune response may be directed to said immunological active part of a microorganism or to a microorganism comprising said immunological active part.

The term “vaccine” as used herein refers to a pharmaceutical composition comprising at least one immunologically active component that induces an immunological response in an animal and possibly, but not necessarily, one or more additional components that enhance the immunological activity of said active component. A vaccine may additionally comprise further components typical to pharmaceutical compositions. The immunologically active component of a vaccine may comprise complete virus particles in either their original form or as attenuated particles in a so-called modified live vaccine (MLV) or particles inactivated by appropriate methods in a so-called killed vaccine (KV). In another form, the immunologically active component of a vaccine may comprise appropriate elements of said organisms (subunit vaccines) whereby these elements are generated either by destroying the whole particle or the growth cultures containing such particles and optionally, subsequent purification steps yielding the desired structure(s), or by synthetic processes including an appropriate manipulation by use of a suitable system based on, for example, bacteria, insects, mammalian or other species, plus optionally subsequent isolation and purification procedures, or by induction of said synthetic processes in the animal needing a vaccine by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination). A vaccine may comprise one or simultaneously more than one of the elements described above. The term “vaccine” as understood herein is a vaccine for veterinary use comprising antigenic substances and is administered for the purpose of inducing a specific and active immunity against a disease provoked by a microbiological infection, such as by a BVDV infection. The BVDV as described herein, confer active immunity that may be transferred passively via maternal antibodies against the immunogens it contains and sometimes also against antigenically related organisms. A vaccine of the invention refers to a vaccine as defined above, wherein one immunologically active component is a BVDV or derived from a nucleotide sequence that is more than 70% homologous to any known BVDV sequence (sense or antisense).

The term “live vaccine” refers to a vaccine comprising a replication competent, in particular, a replication compentent viral active component.

A “pharmaceutical composition” essentially consists of one or more ingredients capable of modifying physiological e.g. immunological functions of the organism it is administered to, or of organisms living in or on the organism. The term includes, but is not restricted to, antibiotics or antiparasitics, as well as other constituents commonly used to achieve certain other objectives like, but not limited to, processing traits, sterility, stability, feasibility to administer the composition via enteral or parenteral routes such as oral, intranasal, intravenous, intramuscular, subcutaneous, intradermal or other suitable route, tolerance after administration, and controlled release properties. One non-limiting example of such a pharmaceutical composition, solely given for demonstration purposes, could be prepared as follows: Cell culture supernatant of an infected cell culture is mixed with a stabilizer (e.g. spermidine and/or BSA (bovine serum albumin)) and the mixture is subsequently lyophilized or dehydrated by other methods. Prior to vaccination, said mixture is then rehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) or non-aqueous solutions (e.g. oil emulsion, aluminum-based adjuvant).

EXAMPLE

The following example serves to further illustrate the present invention; but the same should not be construed as limiting the scope of the invention disclosed herein.

Example 1 Safety of Administering IBR, BVD1, BVD2, PI₃, and BRSV Combination Virus Vaccine to Pregnant Cows and their Calves 1. Summary

The objective of this study was to demonstrate the safety of modified live Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea (BVD) Type 1 and 2 vaccine components as part of booster vaccines for use in previously vaccinated first, second and third trimester pregnant cows and heifers. The study monitored the following primary parameters: Pregnancy outcome from all three trimesters and post-natal health of the calves; Pre-colostral serological status of selected calves from the second and third trimesters.

Cows and heifers from three cow/calf ranches in north-central Nebraska were enrolled to determine calving outcome and calf health status. Cows from a cow/calf ranch in Alberta Canada were also enrolled.

a. Nebraska Sites:

For the Nebraska sites, all cows and heifers were given a pre-breeding vaccination of 10-way vaccine (IBR, BVD 1, BVD 2, PI3, BRSV MLV rehydrated with 5-way Leptospira bacterin) (BREED-BACK™ FP10) one to two months prior to breeding. Booster vaccinations (Test vaccine Group A, 10-way vaccine (BREED-BACK™ FP10), Product Code 4469.23 or Placebo vaccine Group B, 5-way Leptospira bacterin, Product Code 2665.00) were administered to the cows and heifers, with the timing of administration based upon trimester assignment. Pregnancy status was confirmed prior to the booster vaccination, with the pregnant cattle randomly assigned to either Group A or Group B. Cows and heifers located at the Raymond ranch, along with a supplemental set of cows from the A&K ranch, were revaccinated during the first trimester. Cows and heifers located at the A&K ranch were assigned to vaccination during the second trimester. Cows and heifers located at the Williams ranch were assigned to vaccination during the third trimester.

All cows and heifers were followed through calving and the calving outcome recorded for each case. Calving outcome was recorded as either Open (heifer or cow diagnosed as not pregnant, with no calf), Dead (calving resulted in a dead calf not diagnosed as dystocia), or Live (calving resulted in a live calf diagnosed as normal at calving). Calves that died at or immediately after calving due to calving difficulties (dystocia), weather related deaths (frozen in blizzard) or other non-study related causes (calf crushed when stepped on by dam) were removed from the trial. The animals that were removed were not included in the final study results. The Open and Dead cases were combined under the category Fetal Losses.

The resulting calves were observed for four weeks postpartum. The postpartum treatment rates and deaths were recorded.

The results of the study are summarized as follows: For First Trimester Group A, 306 cows/heifers qualified for enrollment and there were 7 fetal losses for a 2.3% loss rate; For First Trimester Group B, 274 cows/heifers qualified for enrollment and there were 6 fetal losses for a 2.2% loss rate. For Second Trimester Group A, 237 cows/heifers qualified for enrollment and there was 1 fetal loss for a 0.4% loss rate; For Second Trimester Group B, 235 cows/heifers qualified for enrollment and there were 3 fetal losses for a 1.3% loss rate. For Third Trimester Group A, 267 cows/heifers qualified for enrollment and there were 5 fetal losses for a 1.9% loss rate; For Third Trimester Group B, 267 cows/heifers qualified for enrollment and there were 6 fetal losses for a 2.2% loss rate.

A full necropsy was performed on all available fetuses (including dystocia, hypothermia, etc. cases). Tissues and body fluid samples, as available, were submitted to the Animal Disease Research & Diagnostic (ADRD) Laboratory, South Dakota State University for analysis. Tests specific for detection of Infectious Bovine Rhinotracheitis (IBR) and Bovine Viral Diarrhea Virus (BVD) were performed. In addition, Rural Technologies Inc. (RTI) performed virus isolations on all submitted tissues. All tests for viral detection and isolation on all fetal tissues were negative.

Heart blood and other body fluids, as available at necropsy, were tested for antibody to IBR, BVD 1 and BVD 2 by the ADRD laboratory. There were a total of six fetuses that tested positive for antibody, however, due to either site practices (forced feeding of colostrum) or testing problems due to sample toxicity/contamination resulting in the lack of confirmation of the test results, these results did not impact the outcome of the study.

Post-calving observations showed that the enrolled calves had low treatment and death rates. Treatments were necessary in the second and third trimester herds due to a mild outbreak of scours. The observations indicated that the majority of cases cleared in 24 to 48 hours. Few post calving deaths occurred. Necropsy results were negative for virus pathogens in the post calving deaths.

b. Alberta Site:

The trial to examine criteria was performed at a cow/calf ranch near Fort Macleod in south central Alberta, Canada. Cows that had been vaccinated pre-breeding were given booster vaccinations in either the second or third trimester. Valid pre-colostral blood samples were successfully taken from a total of 61 calves from the second trimester vaccinated cows and from 59 calves in the third trimester cows. All valid pre-colostral serum samples tested negative for antibody to IBR, BVD 1 and BVD 2. In addition, all serum samples were tested for IBR virus by virus isolation and for BVD 1 and 2 by RT PCR. All serum samples were negative for the presence of virus.

2. Final Results

The results of this study have shown no indication that vaccination of previously vaccinated pregnant cattle in the first, second or third trimester with the modified live IBR and BVD Type 1 and 2 components would result in deleterious effects to either the pregnant cow/heifer or the calves.

3. Introduction

Combination vaccines containing modified live virus (MLV) IBR and modified live BVD Types 1 and 2 have been developed and registered for use in cattle. Efficacy trials have proven the IBR, BVD 1 and BVD 2 combination vaccines (Express™ and Breed-Back FP 5™, Bovine Rhinotracheitis-Viral Diarrhea-Parainfluenza₃-Respiratory Syncytial Virus Vaccine, Modified Live Virus, and combinations; Boehringer Ingelheim Vetmedica) to be effective in preventing BVD Type 1 and 2 persistent fetal infection when administered prior to breeding. Due to safety issues with the use of the IBR and BVD viruses in naive pregnant cattle, these conventionally attenuated virus vaccines have label warnings against the use of the vaccines in pregnant cattle or in calves nursing pregnant cattle. To remove these warnings from the labels for products which contain the same modified live IBR and BVD 1 and BVD 2 components, safety studies were completed in pregnant cattle.

4. Objectives

The objectives of this study were to perform a field safety trial in order to determine the pregnancy outcome in pregnant cows and heifers when vaccinated prior to breeding and subsequently vaccinated in the first, second or third trimester of pregnancy with a product containing the IBR and BVD Types 1 and 2 components of BIVI fetal protection product; to determine the health of the calves born to the enrolled cows and heifers by post-partum observations for a total of four weeks; and determine pre-colostral serological status for IBR, BVD 1, BVD 2 in calves born from cows and heifers vaccinated in the second and third trimester of pregnancy.

The results of this study provides proof of safety of the use of the modified live viral products containing IBR and BVD 1 and BVD 2 components and permit revision of the labeled safety precautions regarding use of the vaccines in previously vaccinated pregnant cattle and in calves nursing pregnant cattle.

5. Materials and Methods

a. Start of Study:

Nebraska:

The study was considered to be officially started on Day 0 for the first trimester herd enrollment pregnancy check/vaccination which was performed on Day 0. However, the pre-breeding vaccinations for the enrolled cattle started on Day −140, with the first vaccinations occurring in the Raymond herd.

Alberta:

The study was considered to be officially started on Day 0 for the vaccination of Group 1, Third Trimester.

b. End of Study:

Nebraska:

The animal portion of the study was completed with the final observation of the calves on Day 295. The laboratory portion of the study ended on Day 399 with receipt of the final BVD serology testing results from Benchmark BioLabs Inc.

Alberta:

The animal portion of the study was completed with the sampling of the final calf on Day 433. The laboratory portion of the tests was completed between Day 487 and Day 517.

6. Study Design

a. General Description:

This large field safety study was designed to determine the safety of the administration of vaccines containing MLV IBR and BVD 1 and 2 components to previously vaccinated pregnant cows and heifers. This trial consisted of two separate segments, the first being a large field safety trial, conducted in Nebraska that investigated calving outcome and post-partum calf health. The second segment, conducted in Alberta, Canada, involved vaccinations of previously vaccinated pregnant cattle in the second and third trimesters, and obtaining pre-colostral serum samples to determine levels of antibody to IBR and BVD 1 and BVD 2. The Nebraska treatment groups are outlined in Tables B, C, and D and the Alberta Canada treatment groups are outlined in Tables E and F.

TABLE B General Trial Design, Nebraska First Trimester MINIMUM NUMBER TREAT- VACCI- MENT 1 TREAT- GROUP SUBGROUP NATED (Pre-Breeding) MENT 2 First Group A MLV 200 IBR-BVD1- IBR-BVD1- Trimester Vaccine BVD2-PI3- BVD2-PI3- Vaccinates BRSV MLV, BRSV MLV, LEPTO 5 LEPTO 5 Group B 200 IBR-BVD1- Lepto 5 Placebo BVD2-PI3- Bacterin Controls BRSV MLV, LEPTO 5

TABLE C General Trial Design, Nebraska Second Trimester MINIMUM NUMBER TREAT- VACCI- MENT 1 TREAT- GROUP SUBGROUP NATED (Pre-Breeding) MENT 2 Second Group A MLV 200 IBR-BVD1- IBR-BVD1- Trimester Vaccine BVD2-PI3- BVD2-PI3- Vaccinates BRSV MLV, BRSV MLV, LEPTO 5 LEPTO 5 Group B 200 IBR-BVD1- Lepto 5 Placebo BVD2-PI3- Bacterin Controls BRSV MLV, LEPTO 5

TABLE D General Trial Design, Nebraska Third Trimester MINIMUM NUMBER TREAT- VACCI- MENT 1 TREAT- GROUP SUBGROUP NATED (Pre-Breeding) MENT 2 Third Group A MLV 200 IBR-BVD1- IBR-BVD1- Trimester Vaccine BVD2-PI3- BVD2-PI3- Vaccinates BRSV MLV, BRSV MLV, LEPTO 5 LEPTO 5 Group B 200 IBR-BVD1- Lepto 5 Placebo BVD2-PI3- Bacterin Controls BRSV MLV, LEPTO 5

TABLE E General Trial Design, Alberta Second Trimester MINIMUM NUMBER TREAT- VACCI- MENT 1 TREAT- GROUP SUBGROUP NATED (Pre-Breeding) MENT 2 Second MLV 65 IBR-BVD1- IBR-BVD1- Trimester Vaccine BVD2-PI3- BVD2-PI3- Vaccinates BRSV MLV BRSV MLV

TABLE F General Trial Design, Alberta Third Trimester MINIMUM NUMBER TREAT- VACCI- MENT 1 TREAT- GROUP SUBGROUP NATED (Pre-Breeding) MENT 2 Third Group A MLV 65 IBR-BVD1- IBR-BVD1- Trimester Vaccine BVD2-PI3- BVD2-PI3- Vaccinates BRSV MLV BRSV MLV

b. Nebraska Sites:

Trial Sites:

Three working cow-calf production ranches located in the rural Ainsworth/Johnstown Nebraska area were identified as having normally low (between 3 to 5%) calving losses. The chosen ranches did not have recent history of either IBR or BVD abortions. With the exception of 82 additional cows enrolled in the first trimester, each trimester consisted of animals from a single herd. Parity of the animals enrolled varied from first calf heifers up to cows with histories of 10 or more pregnancies.

Cows and heifers at the Raymond/Finney Ranch (included Raymond and Walking Y Ranch cows and heifers) were enrolled in the study as first trimester test animals. In addition, a portion of the cows from the A&K herd were also enrolled as first trimester test animals.

Cows and heifers at the A&K Ranch were enrolled in the study as second trimester test animals.

Cows and heifers at the Williams & Williams (W&W) Ranch were enrolled in the study as third trimester test animals.

Animal Selection and Identification:

All heifers and cows enrolled into the study were physically normal, healthy breeding age female Angus or Angus-cross beef cattle. To prevent enrollment of BVD persistently infected (PI) cattle, all heifers in all three herds were ear notched prior to enrollment. Similarly, the calves of the cows of that year available for enrollment were ear notched. The ear notch samples were submitted to the University of Nebraska Veterinary Diagnostic Laboratory (UNVDL), Lincoln, Nebr. for testing by immunohistochemistry. The semen from bulls used for breeding or the semen used for artificial insemination was tested for the presence of BVD by PCR at UNVDL. In addition, all bulls used for breeding were ear notched.

All enrolled heifers and cows were identified using one or two plastic ear tags, a metal Nebraska state ear tag and an electronic identification device (EID). All forms of identification were confirmed each time the animal was handled. Any missing identification was replaced.

A total of two thousand sixty-three (2,063) cows/heifers completed the pre-breeding vaccinations which occurred between Day −140 and Day −67. Six hundred ten (610) cows/heifers were vaccinated for the first trimester (T1). Seven-hundred seven (707) cows/heifers were vaccinated for the second trimester (T2). Seven-hundred forty-six (746) cows/heifers were vaccinated for the third trimester (T3). These animals were bred and pregnancy checked just prior to the trimester enrollment vaccination. The number of animals available for enrollment at the time of trimester vaccinations was reduced from the initial number due to normal expected losses that included non-pregnant (open) animals, lameness, lightning strikes, etc.

Animal Management and Housing:

All three herds were managed according to their normal husbandry practices, with the exception that cows/heifers from Treatment Group A and Treatment Group B were housed as separate groups (no nose to nose contact between Groups A and B) for a minimum of 30 days after the assigned trimester vaccination. In each case, initial housing was located at the home pastures.

In the case of the A&K cows that were assigned to T1, the 82 cows were separated from the remaining A&K herd for 30 days after the T1 vaccination, after which the cows were allowed to rejoin the main A&K cow herd. Once again, at the time of the T2 vaccination, the cows/heifers enrolled in Treatment Group A were isolated from the remainder of the herd for 30 days.

All herds were fed according to their normal herd practices, as appropriate for age, weight, and pregnancy status. All heifers and cows enrolled in the study were pastured according to a rotation schedule determined by each producer throughout the trial. This included movement of the herd to corn stock pastures for the winter months. Details, including area maps for each herd, are filed with the raw data for the study. Feed for the heifers and cows were supplemented with a protein and mineral supplement (cake) and ground hay during the winter months as deemed necessary by each producer. All rations and supplements were pre-approved by the Investigators and Study Monitor.

Daily herd observations were performed starting on Day −123 and continued through Day 280 for T1, Day 276 for T2 and Day 295 for T3 at which time all enrolled calves were at least 30 days of age. Any abnormalities or other observations or comments were recorded in the notes section of the Clinical Assessment Forms. These are included in the raw data files for the study.

Breeding:

T1 and T3 trial sites used bull breeding. For T2, the heifers and majority of the cows were artificially inseminated, followed by the use of clean up bulls.

Enrollment Pregnancy Checks:

The Raymond/Finney heifers for T1 were ultrasounded for verification of pregnancy status on Day 0. Sixty-one (61) heifers were determined to be between 40 to 70 days of gestation. Cows from the Raymond/Finney herd for T1 were ultrasounded for verification of pregnancy status on Day 14 and Day 15. A total of 458 cows were determined to be between 40 to 70 days gestation and eligible for enrollment in the trial.

The A&K cows enrolled as T1 on Day 16 had been ultrasounded on Day −4 and determined to be between 40 and 70 days of gestation.

The A&K heifers for enrollment in T2 were ultrasounded for verification of pregnancy on Day −6. At that time, 95 heifers were determined to be between 40 to 70 days gestation.

The A&K cows were ultrasounded on Days −5, −4, −1, and 1 for verification of pregnancy status. At that time, 476 cows were determined to be between 40 to 70 days gestation. As noted above, 82 of the animals ultrasounded on Day −4 were enrolled as T1 animals on Day 16.

All A&K cows and heifers determined to be pregnant and within the gestation window required for enrollment in the study were palpated to confirm pregnancy status immediately prior to vaccination enrollment on Day 79 for the heifers and on Days 92 and 93 for the cows. Animals determined to be open or to have health issues at that time were not enrolled in the trial.

The W&W heifers for enrollment in T3 were initially ultrasounded on Day 13. At that time, 132 heifers were determined to be between 40 and 70 days gestation.

The W&W cows for enrollment in T3 were initially ultrasounded on Days 28, 29, and 30. At that time, 428 were determined to be between 40 to 70 days gestation.

The W&W cows and heifers determined to be pregnant and within the gestation window required for enrollment in the study were palpated to confirm pregnancy status immediately prior to vaccination enrollment on Day 177 for the heifers and on Days 196 and 197 for the cows. Animals determined to be open or have health issues at that time were not enrolled in the trial.

Randomization:

The T1 Raymond heifers, T1 A&K cows, T2 A&K cows and heifers, and the T3 W&W cows and heifers were assigned to treatment group A or B utilizing randomization software. For each set of vaccinations, two series of random numbers were generated. If the higher random number was listed in the first column of numbers, the assignment was to Group A. If the higher random number was in the second column of numbers, the assignment was to Group B. The heifer or cow was then allotted to the next assigned treatment group on the spreadsheet as the animals were worked through the chute. In the case that all slots on the spreadsheet were not used or additional group assignments were necessary, the last few animals coming through the chute were randomized to group based upon coin toss.

For the T1 cows, these animals were housed in two separate but adjacent pastures. Assignment of the pasture as a group to Treatment A or B was done by coin toss.

Enrollment Vaccinations:

The Test Vaccine (IBR, BVD 1 BVD Type 2, PI3, BRSV, Lepto 5 Serial #MK-270A or MK-270B) was administered to the cows and heifers enrolled in Treatment A. The Placebo Vaccine (Lepto 5 Bacterin, Serial Number 184-089) was administered to the cows and heifers enrolled in Treatment B. Lepto 5 Bacterin contains all of the Leptospira strains, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira Pomona, as are found in the EXPRESS™ and BREED-BACK™ products including, but not limited to EXPRESS™ 10, BREEDBACK™ 10, BREEDBACK™ FP10, and EXPRESS™ FP10. The use of this placebo was a deviation to the protocol. The protocol stated that the placebo would be sterile water.

T1 Vaccinations:

On Days 0, 14, and 15, 61 heifers, 244 cows and 214 cows, respectively, from the Raymond/Finney herd were determined to meet the enrollment criteria for pregnancy status. These heifers and cows were bled and the assigned vaccine product was administered according to the randomization process. On Day 16, 82 additional cows from the A&K herd were administered appropriate vaccine product or placebo based upon the assigned group. The pregnancy checks for the 82 A&K cows were performed on Day −4, which was a deviation to the protocol which stated that the pregnancy checks would be performed within 7 days of the enrollment vaccination. The mean gestation day for T1 enrollment was approximately 55, with an estimated range between 40-75 days gestation.

T2 Vaccinations:

On Days 79, 92, and 93, 90 heifers, 200 cows and 189 cows, respectively, from the A&K herd were determined to meet the enrollment criteria for pregnancy status. These heifers and cows were bled and the assigned vaccine product or placebo was administered according to the randomization process. The mean gestation day for T2 enrollment was approximately 140, with an estimated range between 120-160 days gestation.

T3 Vaccinations:

On Days 177, 196, and 197, 126 heifers, 251 cows and 166 cows, respectively, from the W&W herd were determined to meet the enrollment criteria for pregnancy status. These heifers and cows were bled and the assigned vaccine product or placebo was administered according to the randomization process. The mean gestation day for T3 enrollment was approximately 210, with an estimated range between 195-225 days gestation.

Pregnancy Outcome and Post-Calving Observations:

All enrolled animals were followed through to calving. Calving results were recorded on the Calving Record Form. All trimester enrolled animals diagnosed as pregnant at the trimester vaccination and determined to be open at the end of the trial were counted as a fetal loss. Cows and heifers diagnosed as open were bled at the time of diagnosis and 1 to 4 weeks later. The protocol originally stated that the paired samples would be taken two weeks apart. The serum sample taken at the time of the trimester enrollment vaccination as well as the paired serum samples taken after fetal loss were all tested for levels of serum neutralizing antibody to IBR, BVD Type 1 and BVD Type 2 to determine if serum antibody levels had increased significantly between the first and second of the paired samples, which could indicate a potential fetal loss due to one of the agents.

Deaths at or just after calving diagnosed as due to dystocia (calving difficulties) were removed from the study. Deaths due to other causes unrelated to the study, such as deaths due to hypothermia, dam inflicted injuries or broken limbs were also removed from the study. However, fetuses and calves that died due to dystocia, hypothermia or injuries were subjected to necropsy and testing. The following tissues (if sampling was possible) were collected from the necropsied fetuses: lung, thymus, brain (cerebellum), liver, kidney, spleen, placenta, stomach contents, heart blood and pleural fluid.

The calves were observed daily for four weeks following birth. Any observed health problems or required treatments were recorded.

Diagnostic Testing:

Tissue samples were submitted to a laboratory for testing. Tests performed included general histology, fluorescent antibody (FA) tests specific for IBR and BVD, Leptospira FA, aerobic culture, mycology culture and serum neutralization for IBR, BVD 1 and BVD 2. When obtained, fetal and post-calving ear notch samples were submitted to the laboratory for BVD immunohistochemistry testing.

Tissue samples were also submitted to RTI for performance of virus isolations in tissue culture specific for IBR and BVD.

Serum Neutralization Tests on Adult Serum Samples:

Serum neutralization assays specific for antibody to IBR on blood samples taken from the adult heifers and cows were performed by Benchmark BioLabs, Lincoln, Nebr. according to a standard procedure for serum neutralization. Serum neutralization assays specific for antibody to BVD Type 1 and BVD Type 2 on blood samples taken from the adult heifers and cows were performed by Boehringer Ingelheim Vetmedica Research and Development according to the standard procedure for Serum Neutralization.

Additional Non-Study Related Treatments Administered to the Enrolled Animals:

Additional treatments administered to the trimester enrolled animals included the following Ivomec® Pour-On (Merial), Guardian® Scour Vaccine (Schering-Plough Animal Health), administered to the cows and heifers, and Alpha 7® Clostridial Bacterin (Boehringer Ingelheim Vetmedica) administered to the calves.

c. Alberta Site:

General Design and Sample Collection:

This was a controlled field safety study involving two groups of cross-bred beef cows. The study was designed to determine the safety of the administration of MLV vaccines containing IBR and BVD1 and BVD2 to previously vaccinated pregnant cattle. The two groups of cows were located on a ranch near Fort Macleod, Alberta Canada. One group of cows was vaccinated in trimester three and the second group was vaccinated in trimester two. Each of the trimester groups was considered to be a separate trial, with the data summarized separately. All cows were bred by natural service at pasture.

After confirmation of pregnancy by rectal palpation, a group of one hundred twenty cross-bred beef cows was vaccinated with the modified live 5-way vaccine (IBR, BVD 1, BVD 2, PI3 and BRSV, APHIS product code 1181.24) in trimester three. A second group of one hundred and forty cross-bred beef cows was vaccinated with the 5-way MLV vaccine rehydrated with Lepto bacterin in trimester two. Based on the VSM guideline, it was not necessary to include a placebo group.

At the time of trimester enrollment vaccination of Group 1 (third trimester vaccination), the cows were also vaccinated with an inactivated Rotavirus/Coronavirus/E coli vaccine. This vaccine was administered as a separate shot in a separate site on the cow and had no affect on the outcome of the study. Following vaccination, each group of cows was housed either in a yard, at the ranch or pastured on range land. Each group of cows was observed daily to check for visual signs of fetal loss or other health problems. The investigator maintained a record of outcome of the pregnancy of all vaccinated cows in a calving record book as well as a daily activity calendar log.

Calves from 65 cows in group 1 and from 67 cows in group 2 were bled shortly after birth to obtain a pre-colostrum sample. If the investigator was unsure but suspected that a calf had suckled colostrum, the investigator did obtain a serum sample from the calf for testing and a note was made either in the in the calving record book or in the daily activity calendar. These calves were also indicated in the Investigator's “Research Data” calving/sampling spread sheet record for each study year.

Animal Selection and Identification:

Cattle selected for enrollment were physically normal, healthy breeding age female bovine, either cows or heifers. This herd had no history of infertility and no history of IBR and/or BVD related reproductive problems.

All cattle were identified with a unique number, using a plastic and/or metal ear tags. Calves were assigned a unique identification number and tagged, with a single tag, at birth based on the tag colour and on the sequence of it birth. The calf's tag was linked to the number of its dam in the calving records. In each of groups 1 and 2, all the cows received the same vaccination treatment. All calving events were observed except for the 2 fetal losses in Group 2 listed in “Calving Outcome” in the Results section below. The investigator selected calves born to cows from each group for serum collection based on his confidence that a calf was sampled before it suckled colostrum, with an objective to collect samples from at least 65 calves from each group. If the investigator was unsure that a calf had suckled, this was noted either in the “Remarks” section of the calving record book and/or in the calendar log of daily activities. Those calves were also indicated in the “Research Data” calving/sampling spread sheet record attached to the investigator's report for each study year.

Calving Records:

The investigator maintained a herd book for each of the two groups as well as a daily calendar log containing activities as part of the routine management of the herd. In addition to the identification of the dam, the date of birth and sex of the calf were recorded. These records also contain any notations applicable to herd health. To assist in herd management, a note on calving ease was made using a numeric scale (1 to 4), the calf's birth weight was estimated and the likely sire was also recorded. In the first year's calving season, the herd record book suggests a different, letter-based, scoring system for calving ease, but, for consistency, the numeric scale that had been used in the first year was used in the second year.

The record forms that were attached as a part of the study protocol were not used to record the results of the study. The investigator instead kept written records of the study in calving record books and daily activity logs. This is a deviation to the protocol, however, it did not affect the outcome of the study.

Serum Neutralization and Polymerase Chain Reaction (PCR):

Blood samples were allowed to clot at ambient temperature and then were processed within 36 hours by centrifugation to permit separation of the serum from the clotted blood cells. Aliquots of serum were transferred to three sterile storage tubes. Tubes were labeled and stored at ≦−20° C. until delivered by the study monitor to the testing laboratory. Delivery of a few of the samples to the processing laboratory was delayed approximately 12 hours beyond the 24 hours recommended by the study protocol. This is a deviation to the protocol. This delay did not affect the integrity of the serum samples and did not affect the outcome of the study. Serum neutralization titers for IBR, BVDV Type 1 (challenge virus, Singer) and Type 2 (Challenge virus, NVSL 125c) were determined on all sera at the Animal Health Laboratory, Guelph using a constant virus decreasing serum assay in appropriate cell cultures using <500 Tissue Culture Infective Dose₅₀ (TCID₅₀) challenge virus. The starting dilution was 1:4.

An RT PCR assay for BVD viruses was conducted on pre-colostral serum samples by the Animal Health Laboratory, University of Guelph, Guelph, Ontario. The RT PCR was conducted according to a published procedure. The serum from each calf was tested for gamma-glutamyl transpeptidase (GGT) to help distinguish a calf that had suckled from one that had not. The GGT testing was performed at the Animal Health Laboratory, Ontario Veterinary College, University of Guelph, Guelph, ON using an enzymatic colorimetric method with a Roche/Hitachi 911 Automatic Analyzer.

As there was only one treatment, the investigator and the laboratory technician who separated the serum from blood samples were not blinded to treatment group assignments. The individuals performing the laboratory examinations of post mortem samples and serum samples were unaware of treatment assignments.

d. Vaccine and Placebo Control Articles:

Vaccine and Placebo Control articles used in the trial at the Nebraska trial sites are shown below in Table G. The same serial of test vaccine was used for the pre-breeding vaccination and the trimester enrollment vaccinations.

TABLE G Serial Test Vaccine Number(s) Use Infectious Bovine Rhinotracheitis-Bovine MK-270A- Pre-breeding Viral Diarrhea-Parainfluenza 3-Respiratory 088 or and trimester Syncytial Vaccine, Modified Live Virus, MK-270B- vaccinations Leptospira Canicola-Grippotyphosa- 088 Hardjo-Icterohaemorrhagiae-Pomona Bacterin, APHIS Product Code 4469.23 Leptospira Canicola-Grippotyphosa- 184-087; Trimester Hardjo-Icterohaemorrhagiae-Pomona 184-089; placebo Bacterin, APHIS Product Code 2665.00 184-090; control (refer to Protocol Deviation 2, vaccinations Addendum 3) Vaccine and Placebo Control articles used in the trial at the Alberta trial sites are shown below in Table H.

TABLE H Serial Test Vaccine Number(s) Use Infectious Bovine Rhinotracheitis-Bovine 263-318A Trimester Viral Diarrhea-Parainfluenza 3-Respiratory vaccinations Syncytial Vaccine, Modified Live Virus, Leptospira Canicola-Grippotyphosa- Hardjo-Icterohaemorrhagiae-Pomona Bacterin, APHIS Product Code 1181.24 Sterile Adjuvanted Diluent 807-136 Vaccine Rehydration

e. Amendments, Deviations and Notes to File:

There was a single Amendment to the Study Protocol from the Nebraska site. The Amendment is outlined below in Table I.

TABLE I Amendment Sites # Affected Description Effect on Outcome 1 A&K Describes the decision No effect on the Ranch to use artificial outcome of the insemination for study the A&K ranch

There were a total of 14 Deviations to the Study Protocol from the Nebraska herds and 3 from the Alberta herd. The Deviations are outlined in the table below in Table J.

TABLE J Deviation Herds Effect on # Affected Description Outcome Protocol Deviations 1 A&K first The last two cows for the day were assigned to No negative trimester treatment group based upon coin toss effect 2 All three The decision was made to use Lepto 5 bacterin as No negative Nebraska placebo based on a request by the herd owners effect herds 3 All three The decision was made to obtain baseline blood No negative Nebraska samples at the time of trimester vaccination effect herds 4 Raymond and Minimum gestation accepted for enrollment set to No negative A&K first 45 days estimate based upon ultrasound estimates effect trimester by the Site Investigator (45 days equal approximately 40-50 days) 5 A&K first Range between pregnancy check and enrollment No negative trimester was at 20 days, not the 7 days as stated in the effect protocol 6 First trimester The protocol stated that a single herd would No negative enrollment represent one trimester. A portion of the A&K effect herd was enrolled as 1st trimester, with the remainder of the herd enrolled as 2nd trimester. 7 A&K second Randomizations needed to be adjusted due to No negative trimester enrollment of non-qualified cow as well as to effect enrollment balance the outcome of the total number of cows enrolled as Group A and Group B 8 A&K second Adjustment of the enrollment of animals No negative trimester determined to be at 45 days gestation (40-50 days) effect enrollment at the time of initial pregnancy check 9 Williams 3rd Calves from cows and heifers enrolled in the third No negative trimester trimester were vaccinated with clostridial vaccine effect enrollment to prevent scours 10 All Nebraska First and second blood samples were not obtained No negative herds/all three within the two week time period for a number of effect trimesters the animals that had or were found to be open 11 All Nebraska First and second blood samples were not obtained No negative herds/all three within the two week time period for a number of effect trimesters the animals that had calf loss or were found to be open 12 A&K second The calf from cow #86 died due to dystocia. A No negative trimester blood sample was obtained at calving but was effect inadvertently listed and shipped with the blood samples from the Williams herd vaccinations. Cow #86 was enrolled in Group B, placebo. This sample was not tested. A second sample was not obtained. 13 Raymond Calf 940Y was not submitted for necropsy after Included in first trimester death at approximately 48 hours after birth. Cow fetal losses for was enrolled in Treatment Group A 2^(nd) trimester, Group A 14 Williams Calf 5131 died approximately 2 hours after birth. Included in third trimester The calf was ear notched but was not submitted for fetal losses for necropsy. Heifer 5131 was enrolled in Treatment 3^(rd) trimester, Group A Group A Protocol Deviations Affecting the Alberta Site 1 Ashley Herd, The record forms provided in the appendix of the No negative Alberta study protocol were not used. The investigator effect instead kept written records of the study in a calving record book and a calendar daily record. 2 Ashley Herd, All cows in the 3^(rd) trimester group were No negative Alberta vaccinated with Ecostar 2RC (Novartis Animal effect Health) at the time of trimester enrollment vaccination. 3 Ashley Herd, Some blood samples were held for 36 hours prior No negative Alberta to processing effect

There was a total of 18 Notes to File that were written for the purpose of clarification of parts of the study for the Nebraska sites and 3 Notes to File for the Alberta site, as shown in the table below in Table K.

TABLE K Note to File Herds Effect on Subject Day Affected Description Outcome Notes to File, Nebraska Trial Sites Change of Ear 148 Raymond Replacement of ear tags, list of No negative Tag Numbers first old and new tags for reference effect trimester enrollment Change of Ear 192 A&K second Replacement of ear tags, list of No negative Tag Numbers trimester old and new tags for reference effect heifers Change of Ear 192 Williams Replacement of ear tags, list of No negative Tag Numbers third old and new tags for reference effect trimester heifers Change of Ear 204 Williams Replacement of ear tags, list of No negative Tag Numbers third old and new tags for reference effect trimester Fetal Shipments 232 Williams Shipment of samples from three No negative third fetuses over a weekend effect trimester Duplicate Ear Tag 279 A&K second A few mature cows and heifers No negative Numbers for trimester were inadvertently given the effect Heifers and Cows same ear tag numbers. The tags of mature cows were changed to have the letter M added Change of Ear 289 Raymond At the time of trimester No negative Tag Numbers first enrollment vaccination ear tags effect trimester were changed for 9 cows. The list provided gives a cross reference for pre-breeding vaccination Assignment to 294 A&K first The A&K ranch cows were No negative groups for and second divided into 3 groups A, B, C effect artificial trimester during breeding process. insemination enrollment Administration of 294 All three This note describes the No negative colostrum to non- Nebraska administration of colostrum to effect; caused suckling calves herds weak calves to provide the initial positive results feeding on necropsy serology Administration of 307 Raymond Cows were poured with No negative non-approved first Permectrin CDS Pour-on for effect product for trimester parasite control parasite control ID discrepancies 344 All three Animal identifications were No negative on SDSU herds incorrect on diagnostic laboratory effect laboratory results results. This list provides a cross reference Removal of cow 365 Williams Cow 3028 aborted during chute No negative 3028 prior to third work on Day 197 prior to effect. enrollment trimester enrollment. The fetal samples vaccination were submitted for testing, the results are included in the raw data. Cow was not enrolled Cow 713 365 A&K second Cow number 713 was not No negative pregnancy checks trimester ultrasounded as scheduled but effect was pregnancy checked and confirmed to be pregnant at enrollment vaccination on Day 458 and was enrolled in Treatment Group A Ear notch not 365 A&K second At necropsy, an ear notch was not No negative collected trimester collected from calf 504Y at the effect time of death Study record 379 A&K first The 82 cows enrolled in the first No negative designation for trimester trimester were included in the effect A&K first enrollment data capture forms for the A&K trimester ranch. First trimester cows that enrollment were enrolled were designated with a red dot in the study files Cross-fostered 379 All Nebraska Reference list for calves that No negative calves herds were cross-fostered effect Removal of heifer 379 A&K second Heifer 577 aborted prior to The results 577 prior to trimester enrollment vaccination. The fetus indicated possible enrollment was sampled and tested. The IBR exposure. results are included in the raw Results could not data. Not enrolled in the trial. be confirmed; not enrolled in the trial Randomization of 428 Raymond The Raymond herd was housed No negative Raymond cows, cows, first in two separate groups with effect first trimester trimester approximately equal numbers in each group. A coin toss was used for group assignment Notes to File, Alberta Trial Site Clarification of 394 Alberta 2^(nd) trimester enrollment Cow 190 No negative death loss, Cow Ashley herd (dam of calf 25Y) died of an effect 190 esophageal abscess. Her calf was cross-fostered to cow 216. A serum sample was not taken from this calf Clarification of 392 Alberta 2^(nd) trimester enrollment Cow 208 No negative death loss, Calf Ashley herd gave birth to calf 28Y. This calf effect 28Y was humanely destroyed after its jaw was fractured. A serum sample had been collected from 28Y. Calf 51Y, a co-twin born to Cow 45, was cross-fostered onto cow 208. Calf 51Y was not bled. Clarification of 397 Alberta a 2^(nd) trimester cow calved on No negative notation on “Here Ashley herd Day 397. Her identification is effect BWF” noted as “Here BWF” and her identification calf was identified as 66Y. This cow had lost her plastic ear tag but had been amongst the group confirmed pregnant and vaccinated for the 2^(nd) trimester. Calf 66Y was bled.

7. Results

a. Nebraska Sites:

Ear Notch:

Ear notch results were all negative for both the enrollment testing of calves and heifers and for testing conducted on dead fetuses and calves. The negative results indicate that none of the enrolled cattle were persistently infected and that none of the fetal and calf losses in the trial were due to persistent infection with BVD.

Calving Outcome:

All cows and heifers were followed through calving and the calving outcome recorded for each case. Calving outcome was recorded as either Open (heifer or cow diagnosed as not pregnant, with no calf), Dead (calving resulted in a dead calf not diagnosed as dystocia), or Live (calving resulted in a live calf diagnosed as normal at calving). Calves that died at or immediately after calving due to calving difficulties (dystocia), weather related deaths (frozen in blizzard) or other non-study related causes (calf crushed when stepped on by dam) were removed from the trial. The animals that were removed were not included in the final study results. The Open and Dead cases were combined and included under the category Fetal Losses.

There were no calving losses/abortions that were diagnosed as due to either IBR or BVD. The total number of cows and heifers and a summary of the results that include all possible study related fetal losses from the first trimester are shown in the following tables.

TABLE L Summary of the first trimester calving outcome results Treatment Group Total Calf Losses/Total Enrolled Percent Fetal Losses A 7/306 2.3 B 6/274 2.2 Total 13/580  2.2

TABLE M Summary of the second trimester calving outcome results Treatment Group Total Calf Losses/Total Enrolled Percent Fetal Losses A 1/237 0.4 B 3/235 1.3 Total 4/472 0.8

TABLE N Summary of the third trimester calving outcome results Treatment Group Total Calf Losses/Total Enrolled Percent Fetal Losses A 5/267 1.9 B 6/267 2.2 Total 11/534  2.1

Postpartum Observations:

Calves were observed for 4 weeks postpartum. All calf losses and treatments were recorded. Calves that were lost close to calving were included as a fetal loss and not as a post-calving loss. The results of post-calving observations for all three trimesters showed that the calf losses were low. The treatments consisted primarily of treatments for minor cases of scouring that cleared within 24 to 48 hours.

A summary of the post-calving treatments and post-calving losses for the first trimester are shown in the tables below.

TABLE O Summary of the first trimester postpartum treatments Postpartum Treatments/ Treatment Group Total Enrolled Percent Calf Treatments A 6/299 2.0 B 1/268 0.4 Total 7/567 1.2

TABLE P Summary of the first trimester postpartum calf deaths Postpartum Deaths/ Treatment Group Total Enrolled Percent Calf Deaths A 3/299 1 B 0/268 0.0 Total 3/567 0.5

A summary of the post-calving treatments and post-calving losses for the second trimester are shown in the following tables.

TABLE Q Summary of the second trimester postpartum treatments Postpartum Treatments/ Treatment Group Total Enrolled Percent Calf Treatments A 6/236 2.5 B 2/232 0.9 Total 8/468 1.7

TABLE R Summary of the second trimester postpartum calf deaths Postpartum Deaths/ Treatment Group Total Enrolled Percent Calf Deaths A 0/236 0.0 B 1/232 0.4 Total 1/468 0.2

A summary of the post-calving treatments and post-calving losses for the third trimester are shown in the following tables.

TABLE S Summary of the third trimester postpartum treatments Postpartum Treatments/ Treatment Group Total Enrolled Percent Calf Treatments A 10/262 3.8 B  7/261 2.7 Total 17/523 3.3

TABLE T Summary of the third trimester postpartum calf deaths Postpartum Deaths/ Treatment Group Total Enrolled Percent Calf Deaths A 3/262 1.1 B 1/261 0.4 Total 4/523 0.8

Laboratory Results:

Tissue Samples for Detection of IBR, BVD 1 and BVD 2:

With a few minor exceptions, a full necropsy with tissue sampling was performed on all available fetal and calf losses that occurred during the trial, including dystocia and hypothermia cases.

A necropsy was not performed and samples for testing were not obtained on calves 940Y or 5131 due to failure of the rancher to report the dead calf to the site investigator for necropsy. An ear notch sample was taken from calf 5131 and was reported as negative. Calf 940Y was from a cow enrolled in Group A of the Raymond first trimester herd. Calf 940Y died approximately 48 hours after birth. Calf 5131 was from a heifer enrolled in Group A of the Williams third trimester herd. Calf 5131 died approximately 2 hours after birth.

Animals that died due to a cause obviously unrelated to the study, for example euthanized due to broken leg, were not submitted for necropsy. Tissues and body fluid samples, as available, were submitted to a laboratory for analysis. Tests specific for detection of IBR and BVD were performed by the laboratory. In addition, RTI performed virus isolations on all submitted tissues.

All tests for IBR and BVD 1 and BVD 2 viral detection and isolation on all fetal and calf tissues were negative.

Heart blood and other body fluids, as available at necropsy, were tested for antibody to IBR, BVD 1 and BVD 2 by the laboratory. There was a total of six fetuses or calves that tested positive for antibody to IBR, BVD 1 and/or BVD 2. These are summarized in Table U.

TABLE U Fetuses and calves with reported positive antibody results from necropsy fluid samples Treatment IBR SN BVD 1 SN BVD 2 SN Fetus/Calf # Herd/Trimester Group Diagnosis Titer Titer Titer  577 A&K 2nd/Not Not Aspergillus sp. 1:8 (heart <1:8 <1:8 enrolled Applicable abortion blood)  207Y Raymond/1st A Weak calf, died at <1:4 1:64 (pleural 1:128 24 hours (fed fluid) (pleural colostrum) 1:32(heart fluid) blood) 1:64(heart blood)  86 A&K/2nd B Died, dystocia <1:4 1:16 (heart 1:32 (heart blood) blood) 5128 Williams/3rd A Died, dystocia <1:4 <1:8 1:16 (pleural fluid) 5013 Williams/3rd A Enteritis, had 1:8 1:128 1:512 suckled (pleural (pleural (pleural fluid) fluid) fluid) 5020 Williams/3rd B Euthanized after 1:4 1:64 (pleural 1:32 (pleural birth (pleural fluid) fluid) malformed had fluid) suckled

Since the results on the pleural fluids on the BVD 1 and BVD 2 serum neutralizations for #86 and #5128 were borderline (one to two 2-fold dilutions above negative, with #86, one dilution above negative for only one BVD type), retests were requested to confirm the results. In both cases, attempts at retests were not successful due to the level of toxicity and/or contamination in the sample.

Serum Samples from Cows and Heifers Having Fetal Loss:

Serum samples were obtained from cows and heifers at the time of diagnosis of calf loss or status as open and again at approximately two weeks after the diagnosis. These serum samples as well as the serum sample from the animal that was obtained at enrollment vaccination were tested for the level of serum neutralizing antibody to IBR, BVD 1 and BVD 2. The cow/heifers tested are summarized in Table V below.

TABLE V Serum neutralization results from heifers and cows with calf loss Treatment Animal # Herd/Trimester Group Reason for Sampling 522Y Raymond/First B Stillborn calf 4108 A&K/First A Open 210 A&K/Second B Open 018 A&K/Second B Dead Calf (dystocia) 391 A&K/First B Dead Calf (dystocia) 527 A&K/Second B Dead Calf 593 A&K/Second B Dead Calf, pre-mature 433 A&K/First B Open 220Y Raymond/First A Open 218Y Raymond/First A Dead Calf (dystocia) 243 O Raymond/First B Open  10 O Raymond/First B Open 166Y Raymond/First A Open 300W Raymond/First B Open 516Y Raymond/First B Open 925 A&K/Second A Dead Calf (dystocia) 859 A&K/Second B Open 630 A&K/Second A Open 950Y Raymond/First A Stillborn Calf 426Y Raymond/First A Dead Calf (dystocia) 813Y Raymond/First A Open 945Y Raymond/First A Open 620Y Raymond/First A Open 5127 Williams/Third B Dead Calf (dystocia) 5045 Williams/Third A Dead Calf (dystocia) 5075 Williams/Third B Dead Calf (dystocia) 5109 Williams/Third A Dead Calf (dystocia) 024 Williams/Third B Stillborn Calf 125 Williams/Third B Open 4032 Williams/Third B Open 4040 Williams/Third A Open 341 Williams/Third B Calf died after birth - infected navel 5131 Williams/Third A Dead Calf - Premature 5128 Williams/Third A Dead Calf (dystocia) 947 A&K/Second B Live Calf; died with/in 48 hours 1045 Williams/Third A Live Calf, died bacterial enteritis 2056 Williams/Third B Open 4010 Williams/Third A Stillborn Calf 5012 Williams/Third B Calf died, dystocia 5020 Williams/Third B Euthanized, malformed 5030 Williams/Third B Dead Calf (dystocia) 5086 Williams/Third A Died, stepped on by cow 5103 Williams/Third A Open 577 A&K/Not enrolled Not Aborted Applicable 5041 Williams/Third A Twins, premature 5002 Williams/Third B Euthanized, navel hernia

The serological results for IBR, BVD 1 and BVD 2 were all within the levels that would be expected in a well vaccinated herd. There were a number of cows and heifers that showed a significant rise in titer after vaccination, but none of the animals showed a significant rise between the time of sampling at calf loss and the post-calving blood sampling, which would be indicative of a calf loss due to either IBR or BVD.

b. Statistical Analysis:

Based upon the guidelines published in VS Memorandum 800.110, for each group, the calving rate, calf losses due to unknown causes, and the health status of the calves up to 4 weeks post partum were determined and summarized. The Clopper-Pearson 95% confidence interval for the aborting fractions due to IBR and BVD was calculated for each trimester group. All rate estimates and confidence intervals were calculated for each treatment group and for the groups combined. PROC FREQ of the SAS® System, version 9.1.3, was used to calculate the proportions. The rates (%) were obtained by multiplying the proportions by 100. The Clopper-Pearson confidence intervals were calculated using StatXact from Cytel Studio 7.

Abortion Rate Due to IBRV/BVDV:

For all three trimesters, no cows or heifers (0.0%) in either group were diagnosed as having aborted due to IBRV/BVDV. For the first trimester, the upper 95% confidence limits were 1.2%, 1.3%, and 0.6% respectively for group A, group B, and the groups combined. For the second trimester, the upper 95% confidence limits were 1.5%, 1.6%, and 0.8% respectively for group A, group B, and the groups combined. For the third trimester, the upper 95% confidence limits were 1.4%, 1.4%, and 0.7% respectively for group A, group B, and the groups combined.

Abortion Rate Due to Any Cause:

For all three trimesters, the estimated abortion rate due to any cause was less than 5% for both groups and for the groups combined. For the first trimester, the estimated rate was 2.3% for group A, 2.2% for group B, and 2.2% for the combined groups. For the second trimester, the rate was 0.4% for group A, 1.3% for group B, and 0.8% for the combined groups. For the third trimester, the rate was 1.9% for group A, 2.2% for group B, and 2.1% for the combined groups.

Post-Calving Treatment Rate:

The estimated rate of treatment for any cause for both treatment groups and groups combined was ≦3.8% for all three trimester groups. For the first trimester, the estimated rate was 2.0% for group A, 0.4% for group B, and 1.2% for the groups combined. For the second trimester, the estimated rate was 2.5% for group A, 0.9% for group B, and 1.7% for the groups combined. For the third trimester, the estimated rate was 3.8% for group A, 2.7% for group B, and 3.3% for the groups combined.

Post-Calving Death Rate:

The estimated rate of death by any cause for both treatment groups and groups combined was ≦1.1% for all three trimester groups. For the first trimester, the estimated rate was 1.0% for group A, 0.0% for group B, and 0.5% for the groups combined. For the second trimester, the estimated rate was 0.0% for group A, 0.4% for group B, and 0.2% for the groups combined. For the third trimester, the estimated rate was 1.1% for group A, 0.4% for group B, and 0.8% for the groups combined.

c. Alberta Site Results:

Calving Outcome:

In group one (third trimester), one cow (#119) gave birth to twins. One co-twin was dead at delivery and the second died shortly after birth. Both calves were subjected to a post mortem examination, and had no gross or histopathological or virological evidence of exposure to IBR or BVD viruses. In summary, for group 1, 120 cows were vaccinated with one pregnancy loss. This gives a 0.8% pregnancy loss for group 1, third trimester.

In group two (second trimester), cow 137 died of peritonitis as a sequel to surgical repair of a vaginal prolapse before she had an opportunity to give birth. Cow 115 was observed to abort a fetus, but the fetus had been consumed by wildlife so it was not available for post mortem examination. Three cows (#264, #132, #109) gave birth to calves that were either dead at birth or died shortly thereafter and were classified as stillbirths. All were subjected to a post mortem examination and had no gross or histopathological or virological evidence of exposure to IBR or BVD viruses (see attached post mortem reports Addendum 3). One cow (#216) aborted a fetus that was recovered. It was negative for exposure to IBR or BVD viruses on gross, histopathological and virological examination (see attached post mortem reports Addendum 3). Cow 161 had not calved and was determined to be not pregnant by rectal palpation. The loss of the fetus had not been observed and so it was not possible to conduct a post mortem examination.

In summary, among the 140 enrolled cows that finished the trial in group two, 6 cows either lost a fetus during gestation or gave birth to a stillborn calf. Four of these fetuses and calves were recovered and subjected to a post mortem examination. In each case, the post mortem examination showed no evidence of in utero exposure to IBRV, BVD1 or BVD2. For the remaining two cows, no fetus was available for post mortem examination. This gives a 4.3% pregnancy loss for the second trimester group of cows.

Calf Treatments in First 45 Days of Life:

No calves born in the first or second calving season were treated for illness in their first 45 days of life.

Results on Calves that had Suckled Colostrum:

In group one, the investigator indicated that 5 of the 65 calves had been suspected to have suckled colostrum before a blood sample was collected (calves: 8Y, 16Y, 23R, 25Y, 46R). According to the GGT results, 4 calves (8R, 8Y, 25Y, 46R) had nursed (GGT>51 U/L) and each of these calves were seropositive for IBR, BVD1 and BVD2. These calves were seropositive as a result of having absorbed colostral antibody. Of the four calves that had laboratory tests indicating suckling, three had been identified by the investigator as having nursed. Calves 16Y and 23R, which were suspected to have suckled, were seronegative for IBR, BVD1 and BVD2 and negative for BVDV on RT-PCR and IBRV on virus isolation. If calves 16Y and 23R did nurse, then nursing must have occurred immediately prior to the blood being drawn, with the time interval insufficient for antibody and GGT transfer and elevation. The negative antibody and virus test results indicate that these calves were not exposed to IBR, BVD1 or BVD2. In group two (second trimester), the investigator indicated that 3 of the 67 calves had been suspected to have suckled colostrum before a blood sample was collected (53R, 61R, 72R). Calves 53R and 72R were seronegative to IBR, BVD1 and BVD2 and negative for BVDV on RT-PCR and IBRV on virus isolation. If calves 53R and 72R did nurse, then nursing must have occurred immediately prior to the blood being drawn, with the time interval insufficient for antibody transfer and GGT elevation. The negative antibody and virus isolation results indicate that these calves were not exposed to IBR, BVD1 or BVD2. The investigator indicated that calf 61R was suspected to have suckled. Although the GGT results were below the upper limit of the normal range (51 U/L), the antibody testing indicated that the calf had low virus neutralizing activity against IBR (1:4), BVD1 (1:48) and BVD2 (1:6). Based on the investigator's observations and the test results having low positive antibody for all three viruses, suckling must have occurred very close to when the serum sample for 61R was obtained. Results of IBR virus isolation and BVD 1 and BVD 2 RT PCR testing on the serum from calf 61R were negative. Calf 37R was determined to have nursed unobserved because its GGT value was 2143 U/L (upper limit of normal range: 51 U/L). It was seropositive for IBR, BVD1 and BVD and negative for BVDV on RT-PCR and IBRV on virus isolation.

TABLE W Summary of laboratory results of calves testing seropositive: GGT IBR BVD1 BVD2 BVDV RT- IBRV Calf ID U/L VN VN VN PCR Virus Isolation First Calving Season  8R 1456 1:384 1:2048 1:256 Neg Neg  8Y 746 1:24 1:1024 1:256 Neg Neg 25Y 595 1:16 1:512 1:96 Neg Neg 46R 200 1:4 1:64 1:16 Neg Neg Second Calving Season 61R 26 1:4 1:64 1:6 Neg Neg 37R 2143 >256 >4096 >4096 Neg Neg

Results on Enrolled Calves:

In group two, second trimester, two calves that were otherwise healthy, died by misadventure in the first 30 days of life. Calf 28Y was injured, likely from a cow's kick, causing a broken jaw. It was humanely destroyed and did not have a post mortem examination. Calf 28Y had been identified for blood collection at birth. At birth, calf 28Y was seronegative for IBR, BVD1 and BVD2 and negative for BVDV on RT-PCR and IBRV on virus isolation. Calf #56R was swept down an embankment during a severe rain storm and was not available for post mortem examination. Calf 56R had been identified for blood collection at birth. At birth, calf 56R was seronegative for IBR, BVD1 and BVD2 and negative for BVDV on RT-PCR and IBRV on virus isolation.

In group two, the serum samples from two calves (49R, 98Y) were lost when, on two separate occasions, the centrifuge at the Fort Macleod Veterinary Clinic malfunctioned. The malfunction caused the clotted blood tube to break with complete loss of the serum sample. A total of 65 calves from cows in the third trimester (group 1) were bled shortly after calving. Based upon a combination of investigator observations, antibody testing for IBR and BVD 1 and BVD 2 and GGT testing, the results indicated that four of the calves may have suckled prior to sampling (8R, 8Y, 25Y, 46R). An additional two calves (16Y, 23R) were suspected of having suckled but were seronegative and had normal GGT results. Due to the question on the validity of the pre-colostral serum sampling, all six calves were removed from further consideration in the study. The remaining 59 calves from the third trimester group 1 were all negative for antibody to IBR, BVD 1 and BVD 2. All IBR virus isolations and BVD 1 and BVD 2 RT PCR test results from serum were negative for all calves. There were no post-calving losses due to health problems in this group. A total of 67 calves from cows in the second trimester (group 2) were bled shortly after calving. Based upon a combination of investigator observations, antibody testing for IBR and BVD 1 and BVD 2 and GGT testing, results indicated that two of the calves (37R, 61R) may have suckled prior to sampling. The investigator indicated there was evidence that an additional two calves (53R, 72R) may had suckled. Results of testing from these two calves were negative for both antibody and GGT, indicating that if suckling had occurred, the colostrum was consumed close enough to the blood sampling that passive transfer had not yet occurred. Due to the question of validity of the pre-colostral serum sampling, all four calves were removed from further consideration in the study. Two serum samples were lost due to malfunctions during the processing of the serum; therefore these two calves were also removed from further consideration in the study. The remaining 61 calves from the second trimester group 2 were all negative for antibody to IBR, BVD 1 and BVD 2. The blood samples from all calves were negative for IBR virus isolation and BVD 1 and BVD 2 testing by RT PCR. There were no post calving losses in this group due to health problems.

d. Discussion:

This study was completed in four working cow/calf herds, three located near Ainsworth, Nebraska and a fourth located in Alberta, Canada. Cows and heifers enrolled in the study were vaccinated prior to breeding and at the time of enrollment in the trial with vaccine containing field dose levels of cytopathic modified live IBR and BVD Type 1 and BVD Type 2 components (Boehringer Ingelheim).

For the non-serological Nebraska portion of the trial, data from a total of 580 cattle, 306 vaccinates and 274 placebo controls, were obtained for the first trimester. Data from a total of 472 cattle, 237 vaccinates and 235 placebo controls, were obtained for the second trimester. Data from a total of 534 cattle, 267 vaccinates and 267 controls were obtained for the third trimester.

All enrolled cattle were followed through calving and the calving outcome recorded. Any fetuses or calves recovered from enrolled cattle were subjected to full necropsy that included tests specific to detect IBR and BVD viral agents, both by histological techniques and by virus isolation. There were no positive results in any of the tests that would indicate IBR or BVD was involved in any of the calf losses.

Dystocia, the major cause of new born calf loss, is defined as a difficult or obstructive calving process. For the purposes of this trial, a diagnosis of dystocia as responsible for calf loss was made based upon observations made by the producer and/or Site Veterinarian. The observations were recorded either during calving or at the time of necropsy. Per the agreement made with the Center for Veterinary Biologics prior to the start of the study, calving losses diagnosed as due to dystocia were not included in the overall undiagnosed pregnancy loss rate for the enrolled cattle. After removal of calving losses due to dystocia, the overall undiagnosed calf losses for the cattle enrolled in the trial were below the 5% rate recommended in the guideline. The highest levels were seen in the first trimester group, with 2.3% and 2.2% for the vaccinates and placebo controls respectively. This is to be expected as the enrollment/trimester pregnancy check and vaccination for these cows and heifers was conducted in the first trimester. For the second and third trimester groups, the pregnancy check conducted at the time of enrollment presented the opportunity to eliminate any pregnancy losses that had occurred during the earlier parts of gestation. The level of pregnancy losses in all three trimesters are extremely low and prove the safety of the vaccination of these animals with the modified live vaccine component.

Heart blood and other body fluids, as available at necropsy, were tested for antibody to IBR, BVD 1 and BVD 2 by the laboratory. There were a total of six fetuses that tested positive for antibody, however, due to either site practices (forced feeding of colostrum) or testing problems due to sample toxicity/contamination and the inability to confirm the initial results, these results do not impact the outcome of the study.

Fetus from heifer 577 was reported to have a low levels of antibody to IBR in heart blood. This result was not confirmed. The heifer from the A&K herd aborted prior to the second (gestational) vaccination and was therefore not qualified for enrollment in the trial.

Three calves (207Y Group A 1^(st) trimester; 5013 Group A 3^(rd) trimester and 5020 Group B 3^(rd) trimester) died within approximately 48 hours of birth. Fluids from each of these calves showed relatively high antibody titers to IBR, BVD 1 and BVD 2. However, all three calves had either suckled or were force-fed (tubed) with colostrum prior to death, explaining the positive antibody results.

Fetus from cow 86, Group B, second trimester, died after birth by cesarean section. Death was due to dystocia. The calf presented with hydroperitoneum. Heart blood samples obtained at necropsy were reported as having low titers to BVD 1 and BVD 2. The diagnostic laboratory reported that this sample was found to be toxic to the cell cultures, and therefore the initial results could not be confirmed. The reported problems with toxicity of the serum sample and the inability to confirm the test results indicate that the original results should be considered suspect and, therefore do not impact the outcome of the study.

Fetus from heifer 5128, Group A, third trimester, died at birth due to dystocia. Testing for antibody on pleural fluid initially was reported as having a low titer to BVD 2, and negative for antibody to BVD Type 1. The diagnostic laboratory reported that this sample was found to be toxic to the cell cultures, and therefore the initial results could not be confirmed. The reported low antibody titer and the problems with toxicity of the pleural fluid sample and the inability to confirm the test results indicate that these results should be considered to be suspect and, therefore do not impact the outcome of the study.

In addition, blood samples were taken at the time that the enrollment vaccination (trimester vaccination) was administered. Paired blood samples were also obtained from cows and heifers at the time of diagnosis of pregnancy loss and at an interval of approximately two weeks after diagnosis. Those cattle diagnosed as open, with no recovery of fetal tissue, were of particular interest. The three samples (enrollment vaccination as well as paired samples) were tested to determine the level of antibody to IBR and BVD 1 and BVD 2 at the time of vaccination, at the time of diagnosis of pregnancy loss and at the interval post calf loss.

The serological results for IBR, BVD 1 and BVD 2 in these cattle were as expected for a well vaccinated herd. There were a number of cows and heifers that showed a significant rise in titer after vaccination, but none of the animals showed a significant rise between the time of sampling at the diagnosis of calf loss and the post-calving blood sampling, which would be indicative of a calf loss due to either IBR or BVD.

All calves at the Nebraska sites were followed for 4 weeks post partum. Rates of post partum illness, treatments and deaths in the enrolled calves in all three herds were very low. All three herds experienced minor illnesses that required treatment, most of which were episodes of scouring that persisted for 24 to 48 hours. The highest treatment rates were noted in the Williams herd (third trimester), in which the treatment rate was 3.8% in the vaccinates and 2.7% in the placebo controls. These treatment rates are low for working cow/calf herds.

The post-calving death rates in all three herds were very low and not above the rates that would be expected in normal working cow/calf herds. Necropsy results from all calf deaths were negative for virus pathogens in the post calving deaths.

At the Alberta site, the IBR, BVD 1 and BVD 2 serum neutralizing antibody status in pre-colostral serum samples was determined in a total of 61 calves from the second trimester and 59 calves from the third trimester. Six calves were removed from consideration in both the second and the third trimester due to either equipment malfunction or concerns on the validity of the pre-colostral sample status. The concerns over the validity of the samples were documented by the investigator in the study records. All serum samples from those calves having valid pre-colostral samples were negative for neutralizing antibody to IBR, BVD 1 and BVD 2.

8. CONCLUSION

The results of this study show no indication that vaccination of previously vaccinated pregnant cattle in the first, second or third trimester with the modified live IBR, BVD Type 1 and BVD 2 would result in deleterious effects to either the cow, heifer or the calves.

REFERENCES

The teachings and contents of all references (articles, patents, book portions, presentations, and the like) cited herein, including those listed below, are expressly incorporated by reference herein.

Baker, J. C. 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Med. Assoc. 190: 1449-1458.

Heinz, F. X., Collett, M. S., Purcell., R. H., Cold, E. A., Howard, C. R., Houghton, M., Moormann, R. J. M., Rice, C. M., and Thiel, H.-J. 2000. Family Flaviviridae. PP 859-878. In: Virus Taxonomy (van Regenmortel, H. H. V., Fauquet, C. M., and Bishop, D. H. L, Eds.). Academic Press, San Diego.

Lindenbach, B. D., and Rice, C. M. 2001. The pestiviruses. In Fields Virology, eds. Knipe, Moennig, V. and Plagemann, J. 1992. The pestiviruses. Adv. Virus Res. 41: 53-91.

Thiel, H.-J., Stark, R., Weiland, E., Rümenapf, T. & Meyers, G. 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J. Virol. 65: 4705-4712.31. 

1. A method of preventing abortion in a pregnant cow, comprising administering a first vaccine to the cow prior to pregnancy wherein said first vaccine comprises a therapeutically effective amount of Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhea Virus (BVDV) Type I and BVDV Type 2 and administering a second vaccine to the cow after the cow is pregnant, wherein said second vaccine comprises modified live IBR virus, modified live BVDV Type 1 derived from a Singer strain, modified live BVDV Type 2 derived from Strain 296, and Haemophilus somnus.
 2. The method of claim 1, wherein the modified BVDV Singer strain is the one that is included in the BREED-BACK® FP5 vaccine.
 3. The method of claim 1, wherein the modified live BVDV Type 2 Strain 296 is the one that is included in the BREED-BACK® FP5 vaccine.
 4. The method of claim 1, wherein the modified live IBR is IBR Colorado 1 Strain.
 5. The method of claim 4, wherein the modified live IBR Colorado 1 Strain is the one that is included in the BREED-BACK® FP5 vaccine.
 6. The method of claim 1, wherein the second vaccine further comprises an immunologically active component effective for treatment or prophylaxis of an infection caused by a Leptospira bacterium.
 7. The method of claim 6, wherein the Leptospira bacterium is selected from the group consisting of: Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona.
 8. The method of claim 6, wherein the Leptospira bacterium comprises Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona.
 9. The method of claim 1, wherein the second vaccine is selected from the group consisting of BREED-BACK™ FP5-HS, EXPRESS™ 5-HS®, EXPRESS™ FP5-HS , BREED-BACK™ FP10, EXPRESS™ 10, BREEDBACK™ FP10-HS, EXPRESS FP10-HS®, and combinations thereof.
 10. The method of claim 1, further comprising the step of administering the second vaccine to the pregnant cow during the first, second or third trimester of pregnancy.
 11. The method of claim 1, wherein the second vaccine prevents the incidence of one or more clinical signs of an infection caused by a microbe selected from the group consisting of Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhea Virus (BVDV) Type 1, BVDV Type 2, and any combination thereof in a pregnant cow.
 12. The method of claim 1, wherein the second vaccine prevents the incidence of one or more clinical signs of an infection caused by a microbe selected from the group consisting of Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhea Virus (BVDV) Type 1, BVDV Type 2, and any combination thereof in a fetus of a pregnant cow.
 13. The method of claim 1, wherein the pregnant cow is a pregnant heifer.
 14. The method of claim 1, wherein the modified BVDV Singer strain is the one that is included in the BREED-BACK® FP5 vaccine.
 15. The method of claim 1, wherein the modified live BVDV Type 2 Strain 296 is the one that is included in the BREED-BACK® FP5 vaccine.
 16. The method of claim 1, wherein the modified live IBR is IBR Colorado 1 Strain.
 17. The method of claim 16, wherein the modified live IBR Colorado 1 Strain is the one that is included in the BREED-BACK® FP5 vaccine.
 18. The method of claim 1, wherein the first vaccine further comprises an immunologically active component effective for treatment or prophylaxis of an infection caused by a Leptospira bacterium.
 19. The method of claim 18, wherein the Leptospira bacterium is selected from the group consisting of: Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. 